Project Summary Macrophages are phagocytes of the innate immune system that serve critical roles in host defense, homeostatic processes, and development. Overactive or impaired macrophage activity is associated with the pathology of numerous human diseases, including acute and chronic infections, inflammatory disorders, autoimmune diseases, and metabolic disorders, while the deleterious effects of macrophage insufficiency is evident in primary immune deficiencies such as chronic granulomatous disease. Understanding how macrophages develop and function is a key step toward targeting their activity for therapeutic advantage. ?Primitive? macrophages first appear during the primitive wave in developing embryos, and are thought to function in the pruning of tissues and early innate defenses. These initial macrophages are reinforced by another population of embryonic macrophages that develops from erythromyeloid precursors (EMP), a transient oligopotent precursor that appears prior to the emergence of hematopoietic stem cells (HSCs). It has long been presumed that primitive and EMP-derived macrophages are replaced by HSC-derived macrophages once HSCs are established as the definitive adult hematopoietic precursor. However, several recent reports have challenged this assumption by demonstrating that certain populations of macrophages that reside permanently in tissues originate from a precursor that is seeded prior to birth. Furthermore, these ?tissue- resident macrophages? repopulate themselves through self-renewal and can be maintained independently of HSCs. Despite these advancements, the precise embryonic precursor of tissue-resident macrophages remains unclear due to the overlap of cellular markers between precursor cells and the challenges of in utero experimentation. Since hematopoietic development is highly conserved between vertebrate species, the zebrafish model provides a unique opportunity to circumvent these issues; zebrafish embryos are transparent and develop externally, which makes them especially amenable live imaging and lineage tracing techniques. Furthermore, unlike in mice, zebrafish primitive macrophages, EMPs, and HSCs emerge at different times and in distinct anatomical compartments. In this proposal, lineage-tracing tools to specifically track the fates of primitive and EMP-derived macrophages will be developed and used to compare their persistence in adult tissues. In addition, we will specifically label single cells in situ using the recently described infrared laser gene operator (IR-LEGO) system. This technology will allow us to apply an unprecedented level of targeting precision to map the fate of each precursor. Ultimately, this project will develop a platform to assay potential functional consequences of the divergent origins of tissue-resident macrophages.